Obesity is a serious health problem throughout the world, and contributes to an increased prevalence of Type II diabetes, cardiovascular disease, and cancer. Despite a need for therapeutic options to treat obesity, little is known about the mechanisms that regulate fat storage and release from adipocytes. Triacylglycerols (TAG), the body's major storage form of energy, are packaged in lipid droplets in adipocytes that are covered with perilipin A, a protein that plays a critical role in controlling TAG storage and lipolysis to release fatty acids that serve as a major source of energy. The proposed studies will elucidate the molecular mechanisms by which perilipin A coordinates adipocyte TAG metabolism. Preliminary studies have provided evidence that hormone-sensitive lipase (HSL), and CGI-58, a protein important for TAG metabolism, associate with lipid droplets via a mechanism that is responsive to the phosphorylation state of perilipin A. The proposed studies will test the hypotheses that 1) the phosphorylation of perilipin A controls lipolysis through multiple complementary mechanisms, and 2) that perilipin A serves as an organizing center that coordinates the association of TAG catabolic enzymes with lipid droplets in a manner that is responsive to the lipolytic status of the adipocyte. Specifically, we will 1) elucidate the mechanisms by which perilipin A facilitates the docking of HSL on lipid droplets, 2) identify additional mechanisms by which phosphorylated perilipin A coordinates lipolysis that are distinct from its role in facilitating HSL docking, and 3) elucidate the role that perilipin A plays in docking CGI-58 on lipid droplets and the consequences of this functional interaction to TAG metabolism. Intact and mutated perilipins and HSL or CGI-58 will be expressed in cultured cells that lack these proteins followed by assays for TAG storage and lipolysis; additionally, RNAi approaches and in vitro studies of recombinant proteins are proposed. These studies will contribute to a long-term goal of defining the factors on lipid droplets that control adipocyte TAG metabolism.